Cell cycle regulated repressor and DNA element

ABSTRACT

The present invention relates to a cell cycle regulated repressor protein which binds to a DNA element present in the control sequences of the human cdc25C gene and other cell cycle regulated genes, as well as the use thereof in cell cycle regulated expression systems.

The present invention relates to a cell cycle regulated repressorprotein which binds to a DNA element present in the control sequences ofthe human cdc25C gene and other cell cycle regulated genes, as well asthe use thereof in cell cycle regulated expression systems.

Eukaryotic and prokaryotic cells replicate by a process of cell divisionin which the genome of the cell, be it a single molecule as inprokaryotes or a multiplicity of chromosomes as in eukaryotes, isprecisely replicated before mitosis. Non-dividing, resting cells are ina phase known as G0. When undergoing division, the cell will move in toG1 phase, usually the longest phase, during which the DNA content is 2n(diploid). This is followed by S phase, in which DNA synthesis takesplace and the genome is duplicated. A second G phase follows, G2, inwhich the cell is in a tetraploid (4n) state. Mitosis (M) then occurs,and the cell reverts to G0/G1. The G2→M transition, which involves celland nuclear fission, is controlled by a mitosis promoting factor knownas cdc2, and a cyclin (cyclin B). The human cdc25C gene encodes aprotein phosphatase which activates the cdc2/cyclin B complex prior tothe entry into mitosis.

cdc25C mRNA expression is largely restricted to the G2 phase and isdevelopmentally controlled, but the mechanisms of its regulation havenot been investigated prior to the present study. In fact, G2-specifictranscription has previously not been analysed for any gene in mammaliancells. The molecular mechanisms underlying the periodic induction ofgenes in the G2 phase are therefore unknown.

cdc25 was originally discovered in S. pombe as a cell cycle gene with anessential function in G2→M progression (Russell and Nurse, 1986; for areview see Millar and Russell, 1992). Higher cells contain at least 3genes with a high degree of similarity to cdc25, termed cdc25A, cdc25Band cdc25C, the latter being the closest kin of the S. pombe cdc25(Millar et al., 1991; Nagata et al., 1991; Sadhu et al., 1990). It isnow clear from a number of in vitro studies that the Drosophila,starfish and Xenopus cdc25C genes encode protein phosphatases whichpresumably directly activate the cdc2/cyclin B complex prior to entryinto mitosis (Dunphy and Kumagai, 1991; Gautier et al., 1991; Millar etal., 1991; Sebastian et al., 1993; Strausfeld et al., 1991). In S. pombecdc25 catalysis the dephosphorylation of Tyr-14 in cdc2, therebyreverting the inhibitory action of the wee-1 protein tyrosine kinase(Gould and Nurse, 1989). cdc2 in higher cells is phosphorylated on tworesidues upon formation of a complex with cyclin B, i.e., Tyr-14 andThr-15 (reviewed in Millar and Russell, 1992). As in fission yeast, thisinactivation of cdc2 is mediated by wee-1, which in mammalian cells hasbeen reported to possess tyrosine and threonine kinase activity(Featherstone and Russell, 1991), and both Tyr-14 and Thr-15 aredephosphorylated by cdc25C (Gautier et al., 1991; Kumagai and Dunphy,1991; Strausfeld et al., 1991). These observations demonstrate thatcdc25 and cdc25C play crucial roles during cell cycle progression inmany organisms by triggering the entry into mitosis.

In S. pombe, expression of the cdc25 gene is cell cycle-regulated andcdc25 mRNA and protein reach peak levels during G2 (Ducommun et al.,1990; Moreno et al., 1990). This regulation appears to be of particularrelevance in view of the fact that the level of cdc25, unlike those ofcdc2 or cyclin B, is rate-limiting with respect to entry into M-phase(Ducommun et al., 1990; Edgar and O'Farrell, 1989; Moreno et al., 1990;Russell and Nurse, 1986). In human cells, the level of cdc25C mRNA alsoincreases dramatically in G2, but the abundance of cdc25 protein doesnot vary greatly during the cell cycle (Millar et al., 1991; Sadhu etal., 1990). The same applies to at least one of the cdc25 forms inXenopus oocytes (Jessus and Beach, 1992).

Many diseases, for example cancer, are associated with aberrant cellproliferation.

Cancer is a disorder of the genetic make-up of somatic cells whichresults in a clone of cells with an abnormal pattern of growth control.This leads to unrestricted proliferation of the abnormal clone, whichmay present in the form of a tumour. Available therapy for cancer isbased on the premise that cancer cells, being subject to unrestrictedgrowth, undergo more frequent cell division than normal cells. Agentswhich target dividing cells are therefore seen as useful anti-cancertherapeutics. However, the effectiveness of such agents is limited,since their toxicity to normal cells precludes the administration ofsufficiently effective doses. Moreover, within a tumour cell mass, alarge proportion of the tissue is not rapidly dividing but is in aresting state. Therefore, even if all the dividing cells are eliminated,the tumour clone is not entirely ablated.

A refinement of such techniques which has been proposed is the use ofantibodies or other cell-specific binding agents to target anti-cancerdrugs specifically to tumour cells. For example, reference is made tothe disclosures of EP 0 590 530 and EP 0 501 215 (Behringwerke AG) andreferences cited therein. A difficulty with the proposed techniques isthat it has proven difficult to selectively target cancer cells over thebackground of normal tissue cells from which the cancer has developed,since tumour-specific antigens which are targeted by the antibodies orother binding agents are seldom truly tissue-specific.

Recently, gene therapy techniques have been proposed whereby expressionsystems encoding drugs or enzymes capable of activating prodrugs aretargeted to cancer tissue, and preferably expressed selectively intransformed cells. This method allows the introduction of a furtherlevel of differentiation between tumour and normal tissues, byexploiting tumour-specific expression vectors as well as tumour-specifictargeting systems.

As with antibody delivery systems, however, the drawback with selectiveexpression systems is that background expression levels of theanti-cancer agent encoded by the expression system tend to be excessive,leading to destruction of non-transformed tissue. At the same time, itis difficult to achieve cancer-specific expression using currentlyavailable transcription regulation techniques, since qualitativelycancer cells seem to display very few useful differences from the normaltissue from which they derive.

It has now been found that the cdc25C gene and other cell cycleregulated genes, including cyclin A and cdc2 comprise a DNA elementwhich binds a cell cycle specific repressor factor which, when bound,specifically represses transcription of the linked gene.

According to the first aspect of the invention, therefore, there isprovided a vector for the expression of a desired gene product in acell, comprising a structural gene encoding the desired gene productoperably linked to a promoter under the control of a DNA repressorelement which interacts with a cell cycle specific repressor in order toregulate gene expression in a cell cycle specific manner.

Preferably, the DNA repressor element is derived from a cell cycleregulated gene, such as the cdc25C gene and comprises at least part ofthe sequence 5′-GCTGGCGGAAGGTTTGAATGG-3′ (SEQ ID NO: 1), or afunctionally equivalent mutant or homologue thereof.

Alternatively, the DNA repressor element comprises the sequence5′-GCTGGCGGAAGGTTTGAATGG-3′ (SEQ ID NO: 1) and a sequence encompassing atranscription initiation site, or any functionally equivalent mutants orhomologues thereof.

Preferably, the transcription initiation site is the sequenceencompassing the first major transcription initiation site of the cdc25Cgene.

The DNA repressor element is preferably derived from the cdc25C gene,the cdc2 gene or the cyclin A gene.

The vector of the invention is a nucleic acid vector which may compriseRNA or DNA. The vector may be of linear or circular configuration andadapted for episomal or integrated existence in the target cell. Vectorsbased on viruses, such as retroviral or adenoviral vectors, usuallyintegrate in to the cell genome. Moreover, linear and circular DNAmolecules are known to integrate into cell genomes, as set out in theextensive body of literature known to those skilled in the art whichconcerns transgenic animals.

Where long-term expression of the gene product is sought, integratingvectors are preferred. However, if only transient expression of the geneproduct is sufficient, non-integrating episomes may be used.

The vector of the invention allows the production of a desired geneproduct in a manner which is dependent on the cell cycle phase in whichthe target cell finds itself. For example, in the case of thecdc25C-derived DNA repressor element, the desired gene product will onlybe produced during S and G2 phase, as the target cell prepares formitosis. If desired, therefore, the invention allows production ofspecific gene products in target cells only at specific stages of thecell cycle.

In a particularly preferred embodiment of the invention, the vector isused to encode a cytotoxic agent. Use of such an agent will lead topreferential ablation of cycling cells, which has applications in thetherapy of cancer and other disorders involving aberrant cellproliferation.

The gene product encoded by the vector system may be, in its broadestsense, any polypeptide or ribonucleic acid. For example, the geneproduct may be a polypeptide of therapeutic utility, such as a cytokineor other protein capable of stimulating or modulating an immuneresponse, or a cytotoxic or cytostatic polypeptide. In a preferredembodiment, the polypeptide may be a prodrug-activating enzyme (seeMullen, Pharmac., Ther. 63, 199, (1994)), such as HSV thymidine kinase(TK), capable of converting the non-toxic 6-methoxypurinearabinonucleosides to toxic phosphate derivatives, or cytosinedeaminase, capable of converting 5-fluorocytosine to 5-fluorouracil (seeSikora, K., Tibtech, 11, 197-201, 1993). Other examples includeβ-lactamase, pyroglutamate aminopeptidase, galactosidase orD-aminopeptidase, for example as described in EP 0 382 411 or EP 0 392745, an oxidase such as ethanol oxidase, galactose oxidase, D-amino acidoxidase or α-glyceryl-phosphate oxidase, for example as described in WO91/00108, a peroxidase, for example as described in EP 0 361 908, aphosphatase, for example as described in EP 0 302 473, acarboxypeptidase, for example carboxypeptidase G2 as described in WO88/07378, an amidase or a protease, or most preferably a glucoronidase,especially β-glucoronidase, for example as described in EP 0 590 530.Preferably, the gene encoding the desired gene product encodes a desiredgene product, such as β-glucoronidase, fused to a signal sequence suchas those found in immunoglobulins, to ensure its secretion orlocalisation to the cell surface.

Alternatively, the gene product may be a ribonucleic acid such as anantisense RNA or a therapeutic ribozyme capable of promoting thedestruction of a particular RNA species in a cell. For example,antisense RNA and ribozymes may be targeted to the gene products ofoncogenes. Alternatively, they may be used to ablate specific RNAspecies essential for the survival of the cell, thus acting as acytotoxic or cytostatic agent. Moreover, ribonucleic acids may be usedto target cellular DNA directly, preventing its expression in the cell.

A particularly preferred aspect of the invention is the construction ofa chimaeric promoter that is active preferentially or specifically inthe dividing cells of a specific lineage or tissue. This syntheticpromoter comprises a tissue- or cell type-specific regulatory element inaddition to the repressor element, and should also include the DNAsequence located immediately downstream of the repressor element whichharbours the transcription initiation sites. Preferably, the tissue orcell type-specific regulatory element is positioned upstream of therepressor element. Alternatively, the repressor element may be insertedinto the context of a complete transcription control unit of atissue-specific gene. In order to achieve maximal repression in restingcells it may also be advantageous to insert into the chimaeric promotermultiple copies of the repressor element, preferably in a head-to-tailconfiguration. These strategies provide promoters with dual specificity,i.e. tissue- or cell type-specificity and the dependence on cellproliferation. Where the vector is useful in cancer therapy, thetissue-specific control elements are selected to be active in the tissuefrom which the tumour is derived and to retain their transcriptionactivating function after malignant transformation. Ideally,tissue-specific regulatory elements should not be active in theproliferating, often undifferentiated cells of normal tissue. Thesecriteria are fulfilled by, e.g., the tissue-specific regulatory elementsin the sucrase isomaltase promoter. The combination of such atissue-specific enhancer and a cell cycle-regulated repressor, such asthat of the cdc25C promoter, allows the construction of a vector thatdrives transcription preferentially or specifically in tumour cellsrather than in normal tissue.

The regulatory sequences referred to above include enhancers, which arepreferably tissue specific and contribute to the restriction of theexpression of the gene product to the target cell type. It is anadvantage of the invention that enhancers may be used withoutcompromising the cell cycle regulation of expression from the vector.If, unlike in the present invention, a cell cycle regulated activatorwere used, use of an enhancer would be precluded as this would raise thebackground level of transcription unacceptably. The use of a repressor,however, results in the negation of the effects of the enhancer when thevector is in the repressed state.

Moreover, the regulatory sequences contemplated for use with theinvention include Locus Control Regions (LCRs) as described in EP 0 332667. LCRs have the ability to promote integration-site independentexpression of transgenes and are thus particularly useful where thevector of the invention is to be integrated in to the genome of thetarget cell, as they will ensure that the gene product will beexpressed.

Examples of genes having regulatory sequences, including enhancers,useful for expressing anti-cancer gene products may be found in therelevant literature (e.g. see: Sikora et al., Ann. N.Y. Acad. ofSciences, 716, 115, (1994); Osaki et al., Cancer Res., 54, 5258, (1994);Dihaio et al., Surgery, 116, 205, (1994); and Harris et al., GeneTherapy, 1, 170, (1994). For example, in the case of colonadenocarcinoma, regulatory sequences derived from the carcinoembryonicantigen gene, the sucrase isomaltase gene, the glucagon gene, the villingene or the aminopeptidase N gene may be used; for gastric andoesophageal adenocarcinoma, the sucrase isomaltase gene; for pancreaticcarcinoma, the mucin-1 gene or the villin gene; for small cell lungcarcinoma, the neuron-specific endolase gene or the DOPA decarboxylasegene; for lung adenocarcinoma, the surfactant protein A, B and C genes,the uteroglobulin/CC10 protein gene or the aminopeptidase N gene; forthyroid adenocarcinoma, the calcitonin gene or the thyroglobulin gene;for prostate carcinoma, the prostate-specific antigen gene; and formelanoma, the tyrosinase gene or the TRP-1 gene.

The promoter and control sequences, including the cell cycle regulatedDNA suppressor element of the invention, may be combined with a codingsequence encoding the desired gene product and packaged in a deliverysystem for administration to the target cells. Examples of suitabledelivery systems include viral delivery systems (see Sikora, K.,Tibtech, 11, 197-201, 1993) which, broadly speaking, may be ofretroviral, adenoviral, adeno-associated or any other suitable viralorigin, and include virus-based systems such as virosomes. Moreover,non-viral delivery systems are suitable for use with the invention. Suchdelivery systems include non-targeted delivery systems such asliposomes. However, targeted delivery systems are preferred. Mostpreferred are receptor-ligand mediated uptake systems, particularlyantibody targeted uptake systems. Suitable antibodies for use in suchsystems are particularly antibodies to tumour-associated antigens, suchas antibodies to carcinoembryonic antigen, neural cell adhesionmolecule, the EGF receptor, TAG72, gangliosides GD₂ and GD₃ and otherantigens known in the art. The antibody may be a “natural” antibody suchas IgG, or a synthetic antibody “fragment” such as Fab or Fv, or asingle chain Fv (scFv), which is preferably a humanised antibodyfragment comprising humanised constant regions combined with non-humanCDRs, all of which fragments are described in the relevant literature.

Where the gene product encoded by the vector of the invention is aprodrug activating enzyme, the prodrug may be a cancer-specific prodrug.Alternatively, the prodrug may be an agent which, after activation, hasa general cytotoxic or cytostatic activity. The latter embodiment isparticularly useful in the treatment of tumours.

In a tumour, relatively few of the cells are dividing.

These cells will be ablated by the prodrug on activation. However, ifthe prodrug is capable of killing also a few cells surrounding thetumour cell, non-dividing tumour cells will also be hit, resulting infaster destruction of the tumour. Preferably, the prodrug, onceactivated, is relatively immobile or has a short half-life, such that itwill not be transported in active form too far from the site ofactivation.

In a second aspect of the invention, there is provided a cell cycleregulated transcriptional repressor.

An example of such a repressor is a proteinaceous repressor which, invivo, regulates the expression of a cdc25C gene, preferably of the humancdc25C gene as specifically exemplified herein a cdc2 gene or a cyclin Agene.

The repressor is preferably a protein or a complex of proteins. However,chemical analogues thereof, which are not necessarily proteinaceous butwhich have the same function, are envisaged.

The invention also comprises mutated or otherwise alteredrepressor-derived proteins or chemical analogues of the repressor whichmimic or improve upon its activity. Where the repressor is one of afamily of proteins, the differential activity thereof may be improved oraltered.

The repressor of the invention functions in association with a DNArepressor element which binds specifically to the entity or entitiescomprising the repressor. In the case of the cdc25C repressor, this DNArepressor element is located upstream of, and overlaps, the cdc25C genepromoter and comprises at least part of the sequence5′-GCTGGCGGAAGGTTTGAATGG-3′ (SEQ ID NO: 1), or a functionally equivalentmutant or homologue thereof.

The invention further provides a nucleic acid sequence or sequencesencoding the repressor, as well as an expression vector comprising sucha nucleic acid sequence or sequences.

The nucleic acid sequence or sequences encoding the repressor may encodethe entire repressor or at least one of the components of the repressor,or part of the repressor or part of at least one of the repressorcomponents. Preferably the nucleic acid sequence or sequences are cDNA.

A complementary DNA (cDNA) encoding the repressor or components thereofcan be isolated by screening a mammalian cDNA library constructed in aphage-based prokaryotic expression vector with a radioactively labelledoligonucleotide representing the repressor binding site according toestablished procedures known to those skilled in the art (Singh et al.,1988). The cDNA can be derived from any cell type or tissue where therepressor is expressed, such as human fibroblasts. Alternatively, a cDNAencoding the repressor or components thereof can be obtained byhybridising a mammalian cDNA library constructed in a phage-based vectorto a radioactively labelled, synthetic oligonucleotide probe. This probeis deduced from the amino acid sequence of proteolytic fragments of therespective protein. Such fragments can be obtained by digesting theisolated protein with appropriate proteinases, such as trypsin,chymotrypsin or V8, and separation of the resulting fragments by highpressure liquid chromatography. These techniques as well as themicro-sequencing of polypeptides are described in a vast body ofliterature (e.g., Meyer et al., 1991) and known to those skilled in theart. Isolation of the repressor itself can be achieved by conventionalbiochemical purification procedures (ion exchange, hydrophobic and sizeexclusion chromatography) followed by affinity chromatography using animmobilised, multimeric repressor binding site according to publishedprocedures (Briggs et al., 1986), known to those skilled in the art.

The invention further provides the use of the nucleic acid sequence orsequences, as well as an expression vector comprising such a nucleicacid sequence or sequences, in the production of the repressor protein,protein complex or parts thereof.

It is possible that the repressor will comprise a family of proteins,possibly tissue-specifically expressed, which have differing activitiesin different tissues. In such a situation, the invention comprisesselecting the appropriate protein or proteins from the family in orderto achieve the desired effect in the target tissue.

In a further aspect of the present invention the repressor protein,protein complex or parts thereof can be used in an assay for antagonistsor agonists of the repressor function. Basically, by performing such anassay it is possible to identify substances that affect repressoraction. Methods for identifying such antagonists or agonists are wellknown to those skilled in the art and are described in the body ofliterature known to those skilled in the art relating to such assays.

In a further aspect of the present invention the repressor protein,protein complex or parts thereof and enhancer binding proteins can beused in an assay for antagonists or agonists of the repressor function.Preferably, the enhancer binding proteins interact with the enhancer ofthe cdc25C gene and the equivalent regions of the cdc2 gene or thecyclin A gene. It is further preferred that the enhancer bindingproteins are the glutamine-rich CCAAT-box binding proteins (such asNF-1/CTF) and Sp1 family members. Basically, by performing such assaysit is possible to identify substances that affect repressor action bybinding to either the repressor and/or the enhancer proteins. Methodsfor identifying such antagonists or agonists are well known to thoseskilled in the art and are described in the body of literature known tothose skilled in the art relating to such assays.

The repressor of the invention can be exploited in a variety of ways, toinfluence expression of cell cycle regulated genes and therefore affectthe cycling and growth of cells. This use is of relevance in the controlof disorders and diseases which involve aberrant cell proliferation, asexemplified hereinafter. The repressor may be administered to targetcells using an appropriate delivery system, such as a liposomal deliverysystem, in order to delay or prevent the onset of mitosis.Alternatively, nucleic acid encoding the repressor may be administeredto the cells, again using a suitable delivery system, such as thoseknown to persons skilled in the art and referred to hereinbefore, suchthat the repressor is produced in the target cells in situ.

In a further aspect, the invention provides a method for the treatmentof a disease which is associated with aberrant cell proliferationcomprising the administration to a target cell of a vector according tothe second aspect of the invention, wherein the gene product is oftherapeutic significance in the treatment of the disease.

Diseases amenable to treatment by the method of the invention includecancers of all types, but also other proliferation diseases. Forexample, the treatment of psoriasis is envisaged, as is the treatment ofinflammatory disease, certain viral infections, especiallyvirally-induced cancers and warts, where the virus is responsible forthe deregulation of the cell cycle, fungal infections and proliferativeheart disease.

The invention moreover provides a vector according to the second aspectof the invention for use in medicine.

The invention is illustrated in the appended examples, with reference tothe following figures:

FIGS. 1A and 1B

Cell cycle regulation of cdc25C in the human diploid fibroblast cellline WI-38 after serum stimulation of cells synchronised in G0 by serumdeprivation (FIG. 1A), and in normally cycling HL-60 cells fractionatedby counterflow elutriation (FIG. 1B).

FIG. 1a: Reverse PCR analysis of stimulated WI-38 cells. L7, whoseexpression is not cell cycle dependent, was used as an internal control.Times are intervals post-stimulation. Quantitation of the results was byβ-radiation scanning (Molecular Dynamics PhosphorImager). Relative mRNAcdc25C expression and the fraction of G2 cells as determined by FACSanalysis are plotted against the time post-stimulation.

FIG. 1b: Expression of cdc25C mRNA in elutriated HL-60 cells determinedas in panel a. G: non-fractionated cells; F2 . . . F12: fractionscollected by counterflow elutriation. Quantitation of the results was byβ-radiation scanning and cell cycle analysis as in panel a.

FIG. 2

Nucleotide sequence (SEQ ID NO: 10) of the human cdc25C gene around thetranscription start sites. The two major sites of transcriptioninitiation are marked by a solid square (see also FIG. 3). Protected Gresidues detected by in vivo footprinting (see FIG. 5) are marked byfilled circles (constitutive binding sites 1, 2 and 3: CBS 1, 2 and 3)or triangles (cell cycle dependent element: CDE). The 5′ ends of the C74promoter construct used in FIGS. 4B, 5 and 7 are shown by arrowspointing to the right; the common 3′ end of all deletion constructs isindicated by an arrow pointing to the left.

FIG. 3

Mapping of the 5′ end of cdc25C mRNA by primer extension in normallycycling WI-38 cells (leftmost lane). Control: E. coli tRNA (negativecontrol) A sequencing reaction was run alongside to be able toaccurately determine the 5′ ends of cdc25C MRNA. The two major sites oftranscription initiation are indicated by arrows.

FIGS. 4A and 4B

Kinetics of induction after serum stimulation of quiescent NIH3T3 cellsof different promoter-luciferase constructs: 5×TRE-tk (“TRE”), 973 bp ofthe human cyclin D1 promoter (“Δ973”: Herber et al., 1994) and a 605 bphuman cdc25C upstream fragment in FIG. 4A, and of different truncatedcdc25C promoter constructs in FIG. 4B, which also shows the resultsobtained with the HSV tk-promoter for comparison. Panel A gives themeasured activities (RLUs), panel B shows the level of induction foreach construct tested (i.e., values relative to activity in G₀ cells).

FIGS. 5A and 5B

Transient expression analysis of terminally truncated cdc25Cpromoter-luciferase constructs in quiescent (G₀) versus stimulated (G₂)NIH3T3 cells (FIG. 5A), and in quiescent (G₀) versus normally cycling(growing) cells (FIG. 5B). The stimulated cells were analysed 26 hpost-stimulation, i.e. the majority of these cells were in G₂.

Experiments were performed 4-times with 2 independently prepared sets ofplasmids. Relative activities of the different deletion constructsshowed standard deviations in the range of 5-30%. Mean values andstandard deviations are not indicated in this Figure. to be able to showthe actual luciferase activities rather than normalised values.

FIG. 6A and 6B

Identification of protein binding sites in WI-38 cells by in vivo DMSfootprinting of a region spanning nucleotides −90 and +20 in FIG. 6A andnucleotides −21 and +8 in FIG. 6 B. One G₂-specific binding site (CDE)(located within SEQ ID NO: 22) and 3 constitutive binding sites (CBS 1,2 and 3), which are protected under all growth conditions, can beidentified. All protected G residues are marked in the sequence shown inFIG. 2. Control: naked DNA methylated in vitro. A: Quiescent, stimulatedand growing WI-38 cells. The stimulated cells were 24 hpost-stimulation, i.e. the majority of these cells were in G₂. B:Independent repetition of the experiment in panel A plus G₁ and G₂ cellsisolated by FACS from populations of normally cycling WI-38 cells.

FIGS. 7A and 7B

Transient expression analysis of a cdc25C promoter-luciferase constructcontaining a mutated CDE (construct C74R1) in quiescent (G₀) versusstimulated (G₂) NIH3T3 cells (FIG. 7A, left graph) and in quiescent (G₀)versus growing cells (panel A, right graph). The CDE sequence wasmutated as follows: . . . CTG GCGGAA . . . → . . . CTGATCAAA . . .(protected G residues underlined; mutated bases double-underlined). FIG.7B shows the results obtained in 6 independent experiments. Valuesseparated by slashes indicate luciferase activities obtained with C74(left value) and V74Rl (right value), respectively, under differentgrowth conditions (top panel: G₀ and growing; bottom panel: G₀ and G₂).The increase in promoter activity caused by the CDE mutation is alsoindicated for each pair of values (fold increase). Averages and standarddeviations for G₀ and G₂/growing cells are shown at the bottom,indicating that mutation of the CDE in C74Rl led to an average 12.8-foldincrease in G₀ cells, but only to 1.6-fold increase in G₂/growing cells.

FIGS. 8A and 8B

FIG. 8A) Nucleotide sequence (SEQ ID NO: 11) of the cdc25C upstreamregion. The two major sites of transcription initiation are marked by asolid square. Protected G residues detected by in vivo footprinting aremarked by filled (•) and open (∘) circles to denote strong and partialconstitutive protection, respectively. Cell cycle-regulated proteinbinding to the CDE is indicated by asterisks. Y_(c)-boxes 1, 2 and 3 areshaded, Y_(c)-box 1 being the most downstream one. Arrows show the 5′end points of the deletion constructs used in subsequent Figures. FIG.8B) Alignment of Y_(c)-boxes 1 (SEQ ID NO: 25), 2 (SEQ ID NO: 24) and 3(SEQ ID NO: 23). Filled and open circles indicate G residues that showstrong (•) or partial (∘) protection in all three sequences.

FIG. 9

Transient expression analysis of terminally truncated cdc25Cpromoter-luciferase constructs in quiescent (G₀) versus growing NIH3T3cells. Plasmids were named to indicate the 5′-truncation (see also FIG.8). All plasmids harbour a 121 bp region downstream of the firstinitiation site. Mean normalised values (C290 in growing cells=100%) of3 independent experiments and standard deviations are given. Factor isthe ratio of the values in growing and G₀ cells. Δ Site indicates whichsite was deleted from a given construct with respect to the precedingone (one line above). Arrow heads point to those sites whose deletionled to a significant drop in activity (≧30%).

FIGS. 10A and 10B

Transient expression analysis in quiescent (G₀) and growing NIH3T3 cellsof cdc25C promoter-luciferase constructs harbouring specific mutationsin CBS elements. Black boxes: CBS elements in Y_(c)-boxes; grey boxes:other non-mutated elements; open boxes: mutated CBS elements. Theanalysis and evaluation was performed as in FIG. 9.

FIG. 11

Transient expression analysis in quiescent (G₀) and growing NIH3T3 cellsof various cdc25C enhancer fragments linked to the C20 basal promoterconstruct with either a wild-type or a point-mutated (RT7 constructs)CDE. Black boxes: CBS elements in Y_(c)-boxes; grey boxes: othernon-mutated elements; open boxes: mutated CDE (RT7 mutants). Theanalysis and evaluation was performed as in FIG. 10.

FIG. 12

Mutagenesis of the CDE. Constructs were tested in both quiescent andgrowing NIH3T3 cells.

FIG. 13

Mutagenesis of the region between the CDE and position +30. Constructswere tested in both quiescent and growing NIH3T3 cells. φ: nosignificant difference in activity of wt and mutant form in quiescentcells; +++: 3- to 10-fold deregulation; arrow: −2-fold decreasedactivity. No major differences were seen in growing cells.

FIG. 14

Repression of the SV40 early promoter/enhancer region by the CDE intransient luciferase assays. SV-TATA: A natural SV40 constructcontaining the SV40 early promoter/enhancer region, TATA-box andtranscription start site. SV-C20: fusion construct consisting of theSV40 early promoter/enhancer region linked to a minimal cdc25C promoterfragment (−20 to +121) harbouring a wild-type CDE. SV-C20R1: same asSV-C20, but with a mutated CDE. All constructs were tested in quiescent(G₀) and growing cells. Data were normalised to 100 for SV-TATA in G₀cells.

FIG. 15

Similarities of cell cycle-regulated promoters in the region of the CDEand CHR. cdc25C (SEQ ID NO: 12), cdc2 (SEQ ID NO13), Cyclin A (SEQ NO:14), Cyclin F (SEQ ID NO: 15).

FIG. 16

Similarities of the cdc25C (SEQ ID NOS: 16 and 19), cdc2 (SEQ ID NOS: 17and 20) and cyclin A (SEQ ID NOS: 18 and 20) promoters in the region ofthe CDE-CHR elements and upstream sequences resembling reverse CCAATboxes (Y-boxes 1 and 2 in the cdc25C gene).

MATERIALS AND METHODS Library Screening

A genomic library of the human lung fibroblast cell line WI-38constructed in 1-Fix (Stratagene) was screened with a ³²P-labelled cdc25CDNA probe (Sadhu et al., 1990) cloned by reverse PCR. Hybridisation wascarried out for 24 h at 60° C. in 5×SSC, 0.1% SDS, 5×Denhardts solution,50 mM sodium phosphate buffer pH 6.8, lmM sodium phosphate and 200 μgsalmon sperm DNA per ml. Filters (Pall Biodyne A) were washed at thesame temperature in 0.1% SDS and 0.1% SSC.

Primer Extension Analysis

32P-labelled primer (10 pmol) and total RNA from HeLa cells weredenatured for 10 min at 65 ° C. and then incubated at 370° C. for 30min. Primer extension was carried out in a total volume of 50 μlcontaining 50 mM Tris pH 8.3, 75 mM KCl, 10 mM dithiothreitol, 3 mMMgCl₂, 400 μM dNTPs, 2 U RNasin and 400 U M-MuLV reverse transcriptase(Gibco-BRL). After incubation for 1 hr at 37° C., the reaction wasstopped with EDTA followed by an RNase treatment. The precipitated DNAwas subsequently electrophoresed on a 6% acrylamide/7M urea gel. Thefollowing 5′ primer was used:

5′-CCCCTCGAGGTCAACTAGATTGCAGC-3′ (SEQ ID NO: 2).

Exonuclease III Treatment

For sequence analysis, 5′ deletions of a genomic Accl/EcoR1 fragmentwere performed by exonuclease III digestion using a nested deletion kit(Pharmacia-LKB).

PCR Mutagenesis

Site directed mutagenesis was performed as described (Good and Nazar,1992) with slight modifications. Two complementary primers carrying themutation and an additional restriction site plus a second set of primersfor subcloning were designed. The first PCR reaction (Saiki et al.,1988) was performed with (i) 5′cdc25 and 3′mCDE and (ii) 3′cdc25 and5′mCDE as the primers. The resulting products were purified (QIAquickspin PCR purification; Diagen), digested with the enzyme for the newlycreated restriction site, ligated and amplified in a second PCR reactionusing 5′cdc25 and 3′cdc25 as primers. The resulting fragments carryingthe mutation were cloned into the corresponding restriction sites of thecdc25 promoter-luciferase construct. The mutant was verified bysequencing. The primers had the following sequences:

5′cdc25, 5′-CGCCCCAACACTTGCCACGCCGGCAGC-3′ (SEQ ID NO: 3); 3′cdc25′,

5′-CCCCTCGAGGTCAACTAGATTGCAGC-3′ (SEQ ID NO: 4); 5′MCDE,

5′-GGTTACTGGGCTGATCAAAGGTTTGAATGG-3′ (SEQ ID NO: 5);

3′mCDE, 5′-CCATTCAAACCTTTGATCAGCCCAGTAACC-3′ (SEQ ID NO: 6).

Reverse Transcriptase PCR

For cDNA synthesis, 4 μg of total RNA (Belyavsky et al., 1989) wereannealed to 1 μg of oligo(dT) and incubated with 200 U of M-MuLV reversetranscriptase for 1 h at 37° C. in a final volume of 20 μl. One tenth ofthe reaction mixture was amplified by 17-25 cycles of PCR (Saiki et al.,1988) in the presence of 0.5 μCi α-³²P-dCTP. The PCR products werequantitated by β-radiation scanning using a PhosphorImager (MolecularDynamics).

Cell Culture and DNA Transfection

WI-38 cells were obtained from the ATCC. All cells were cultured inDulbecco-Vogt modified Eagle medium supplemented with 10% fetal calfserum (FCS), penicillin (100 Uml⁻¹) and streptomycin (100 Uml⁻¹). NIH3T3cells were transfected using the calcium phosphate technique. 1×10⁵cells/dish (3 cm diameter) were plated 24 hrs prior to the transfectionof 5 μg of DNA. Cells were harvested by scraping and lysed by threefreeze-thaw cycles. For serum stimulation, cells were maintained inserum free medium for 3 days. Stimulation was carried out for theindicated times with 10% FCS. Luciferase activity was determined asdescribed (Heiber et al., 1992). Results were corrected for transfectionefficiency as described (Abken, 1992).

Genomic Footprinting

For genomic footprinting (Pfeifer et al., 1989), WI-38 cells were grownto 70% confluency. The cells were treated with 0.2% dimethyl sulfate(DMS) for 2 min. After DMS treatment, cells were washed three times withcold PBS, and the DNA was isolated. As a reference, WI-38 genomic DNAwas methylated in vitro with 0.2% DMS for 10-30 seconds. Piperidinecleavage was performed as described. For FACS analysis and sorting, thecells were trypsinised after DMS treatment, resuspended in PBS and fixedin 70% ethanol overnight at 4° C. The fixed cells were washed twice withcold PBS and resuspended in 5 ml DNA staining buffer (100 mM Tris pH7.4, 154 mM NaCl, 1 mM CaCl₂, 0.5 mM MgCl₂, 0.1% NP40, 0.2% BSA, 2 μg/mlHoechst 33258). Cell sorting was performed with a Becton-DickinsonFACStar Plus at a rate of 500-1000 cell/sec. The sorted G1 and G2 cellpopulations were ˜90% and ˜80% pure, respectively. The genomic DNA fromsorted cells was purified on anion exchange columns (QIAamp, Qiagen). 3μg genomic DNA were used for ligation mediated PCR (LMPCR) as described.The Stoffel fragment of Taq polymerase (Perkin Elmer) was used insteadof the native enzyme.

Samples were phenol extracted and ethanol-precipitated before primerextension with ³²P-labelled primers. The following oligonucleotides wereused as primers: LMPCR 1 (this Figure): 1st primer, Tm=56.0° C.,5′-d(AGGGAAAGGAGGTAGTT)-3′ (SEQ ID NO: 7); 2nd primer, Tm=74.0° C.,5′-d(TAGATTG CAGCTATGCCTTCCGAC)-3′ (SEQ ID NO: 8); 3rd primer, Tm=83.0°C., 5′-d(CCTTCCGACTGGGTAG GCCAACGTCG)-3′ (SEQ ID NO: 9).

Results Induction of cdc25C mRNA Expression in G2 in Both Stimulated andNormally Cycling Cells

The cdc25 gene has previously been shown to be expressed specifically inthe G2 phase in HeLa cells. In order to investigate whether the G2specific expression of cdc25 mRNA is a general phenomenon in human cellswe analysed both WI-38 cells synchronised by serum deprivation andstimulation, and normally cycling HL-60 cells fractionated bycounter-flow elutriation. cdc25C RNA levels were quantitated by reversePCR and cell cycle progression was determined by FACS analysis. Theresults of this study (FIG. 1) show that in both cell types and underboth experimental conditions expression of cdc25c RNA was clearly G2specific. Thus WI-38 cells began to enter G2 approximately 20 hrspost-stimulation which coincides with the time when the level cdc25C PNAincreased. Similarly, the fraction of G₂/M cells in different samples ofHL-60 cells obtained by counter flow elutriation closely correlated withthe expression of cdc25C RNA. The induction cdc25C was approximately50-fold in stimulated WI-38 cells and about 10-fold in elutriated HL-60cells. The higher induction in the stimulated WI-38 cells is presumablydue to a lower basal level in quiescent versus G1 cells.

Structure and Function of the Human cdc25C Gene 5′ Flanking Sequence

A genomic library of WI-38 cells was screened with a cDNA clonerepresenting the human cdc25C coding region. A recombinant phagecontaining a 15 kb insert was identified and used for further analysis.The nucleotide sequence of approximately 1800 bp of the 5′ flankingregion was determined for both strands. The most relevant part of thesequence, as determined below, is shown in FIG. 2. To identify thepoint(s) of transcription initiation the 5′ ends of cdc25C mRNA weredetermined by primer extension analysis (FIG. 3). This experiment led tothe identification of two major start points located 227 and 269 bp 5′to the ATG start codon. Since the cdc25C gene is expressed in G₀/G₁ atextremely low levels (see FIG. 1), it was not feasible to analyse apotential cell cycle dependent usage of the two start sites. Inspectionof the nucleotide sequence 5′ to the start sites showed no canonicalTATA box or TATA-like sequence, indicating that cdc25C is a TATA-lessgene.

A cdc25C gene fragment spanning nucleotides −605 to +121 was linked tothe bacterial luciferase gene (construct C605) and transfected intoNIH3T3 cells to test whether the isolated promoter fragment wasfunctional in a transient expression assay. Transfections were performedwith relatively dense cultures which proved to be advantageous for tworeasons: (i) The cells became quiescent more rapidly and efficientlycompared with sparser cultures, and (ii) the protein content inquiescent, cycling and stimulated cells varied by a factor of ≦1.5 (datanot shown), so that it was possible to correlate the measured luciferaseactivity directly to the number of transfected cells (the results wereexpressed as RLUs/2×10⁵ recipient cells). Transfection efficiencies weremonitored by determining the number of plasmids taken up by the cells(Abken 1992), but in general fluctuations were <1.5-fold (not shown). Weprefer this experimental design over the cotransfection of a secondreporter plasmid as an internal standard, because using the formerapproach we avoid complications with respect to serum stimulation of thereporter construct used for standardisation (which is seen to someextent even with promoters like RSV-LTR or SV40).

As shown in FIG. 4A, construct C605 was cell cycle regulated in serumstimulated cells that had been synchronised in G₀. Thus, hardly anyluciferase activity was detectable in G₀ cells and during the first 15hours post-stimulation, i.e. during G1 and early/mid-S, but there was astrong induction at 20 and 28 hrs. after serum stimulation, when mostcells had entered, or passed through, G₂. In the same experiment weincluded two other reporter constructs containing either 5 copies of thehuman collagenase TRE linked to the HSV-tk promoter (Angel et al., 1987)or a 973 bp fragment of the human cyclin D1 promoter (Herber et al.,1994). Both the activation of AP-1 and the induction of cyclin D1transcription are early events following serum stimulation, occurringprior to S-phase entry (e.g. Kovary and Bravo 1992; Sewing et al.,1993). This feature of the endogenous genes is reflected by thetransient assay in FIG. 4A. Both the TRE and cyclin D1 promoterconstructs gave rise to peak luciferase activities at approximately 7hrs., i.e. prior to S-phase entry of the majority of the cellpopulation. These data demonstrate that the transient expression assayclosely mirrors the physiological regulation of the cdc25C gene and issufficient to confer on a luciferase reporter gene a pattern of cellcycle regulation that is similar to that of the endogenous gene.

The level of C605 induction was −50-fold in the experiment shown in FIG.4A, but showed some variation in different experiments in the range of−10- to 50-fold. Likewise, the level of luciferase activity in quiescentcells varied among different experiments (see FIGS. 5A and B). Thesevariations, which are presumably due to unknown factors affecting thecondition of the recipient cells at the time of transfection, havehowever no bearing on the interpretation of the results obtained in thepresent study, since relative activities comparing different constructs(e.g., terminal cdc25C promoter deletions, see below) within a givenexperiment were always very similar with variations <30%.

Identification of a 74 bp Promoter Fragment Conferring Cell CycleRegulation

In order to identify functionally relevant regions in the cdc25Cpromoter we generated a series of terminal truncations and analysedthese for expression in cells synchronised in G₀ versus stimulated cellsin G₂ (FIG. 5A), and in G₀ versus normally cycling cells (FIG. 5B). Inaddition, two longer promoter fragments than the one analysed in FIG. 4Awere analysed. The data in FIG. 5 shows that all constructs apart fromC20, which contains just 20 nucleotides of upstream sequence, wereclearly cell cycle-regulated, in both stimulated and normally cyclingcells. This suggests that the sequence upstream of nucleotide −74 onlyplays a minor role, if any, in cell cycle regulation. This conclusion isstrongly supported by the data in FIG. 4B which show that the inductionkinetics of the four constructs tested, including C74, are very similar.The fact that even C74 showed the expected cell cycle-dependentexpression pattern indicates that the region from −74 to +121 issufficient for late S/G2-specific transcription.

The data in FIG. 5B point to 2 additional regions in the promoter thatseem to play a role in transcription. One is located far upstream (−835to −1067) and its deletion leads to an increased activity, but themolecular basis underlying this effect is not clear. The other region islocated at −74 to −169 and seems to contain a cell cycle-independentenhancer, since its deletion leads to a −5-fold drop in activity in bothG₀ and G₂ cells. The late S/G₂-specific transcription of the humancdc25C gene is therefore dependent on a DNA repressor element and thesurrounding sequences. We have shown that the upstream sequences harbourmultiple in vivo protein binding sites (FIG. 8) which interact withconstitutive transcriptional activators (FIGS. 9-11). Major determinantsin this enhancer region are a bona fide Sp1 site and three directsequence repeats (Y_(c)-boxes; see FIG. 8B) each consisting of anelement resembling a reverse CCAAT-box and an adjacent GC-rich motif. Asindicated by genomic footprinting, functional analyses and in vitroprotein binding studies (Barberis et al., 1987) and antibody supershifts(Mantovani et al., 1992) using nuclear extracts from HeLa cells (Dignamet al., 1983), the Y_(c)-boxes represent unusual binding sites for theCCAAT-box binding protein NF-Y/CBF (Dorn et al., 1987: van Hujisduifnenet al., 1990; Maity et al., 1983) and synergise in transcriptionalactivation.

Efficient transcription of the cdc25C promoter constructs was also seenin normally growing cells, even though only a relatively small fractionof the cell population is in G2 at any given time. We attribute this tothe fact that the luciferase protein is relatively stable and could thusaccumulate to high levels in the growing cells.

Identification of a Cell Cycle-regulated Protein Binding Site in vivo(CDE)

To analyse the mechanisms involved in cell cycle regulation of thecdc25C gene in detail we performed genomic dimethyl sulfate (DMS)footprint analysis of the region between nucleotides −80 and +15 usingG₀ and stimulated (G₂) WI-38 cells, i.e. conditions of minimum andmaximum expression of endogenous cdc25C (see FIGS. 1 and 4). FIG. 6Ashows that within this region guanine residues in 4 distinct areas wereprotected from methylation by DMS. Protection of 3 sites was found to beconstitutive, i.e. independent of the cell cycle. These sites, locatedat positions −28/−27,−39 and −58/−59 were termed constitutive bindingsites 1, 2 and 3 (CBS 1, 2 and 3). Further footprint analysis of theregion between nucleotides −310 and +65 has been made and a further 6CBSs identified. See FIG. 8. Please note CBS 1 and 2, CBS 3 and 4, andCBS 5 and 6 are located within Y_(c)-boxes 1 to 3, respectively. Thesite identified by in vivo footprinting, located at positions −12 to−16, is of particular interest because protein binding to this sequenceis cell cycle dependent (FIG. 5A). Thus, hardly any protection was seenin stimulated cells in G₂. whereas a clear protection was observed inboth quiescent and normally cycling cells. This site was thereforetermed cell cycle dependent element (CDE). The fact that no differencewas seen among quiescent and growing cells is presumably due to the factthat the fraction of G₂ cells in normally cycling cells is relativelylow (10-15%).

In order to rule out any artefacts arising from the synchronisationprocedure, and to analyse whether the CDE footprint was also seen in G₁cells rather than being G₀-specific, we repeated the experiment and thistime included purified G₁ and G₂ cells obtained by preparative cellsorting of a normally cycling population of WI-38 cells using afluorescence activating cell sorter (FACS). The results of thisexperiment are shown in FIG. 6B: four G residues within the CDE atpositions −12, −13, −15 and −16 were protected in G₁ but not in G₂. Wetherefore conclude that protection of the CDE is seen in both G₀ and G₁cells, indicating cell cycle regulated protein binding to the CDE.

The CDE is a Major Determinant of Cell Cycle Regulation of cdc25CTranscription in vivo

In view of the cell cycle regulation seen with Δ74 (FIGS. 4B and 5), theresults obtained by in vivo footprinting (FIGS. 6 and 8) and thetransient expression analysis (FIGS. 2, 3 and 4), it can be seen thatthe CDE and the constitutive binding sites (CBS) 1 to 8 play a crucialrole in cell cycle dependent activation of the cdc25C promoter. Sincethe CDE as a cell cycle regulated protein binding site was aparticularly interesting candidate in this respect, we generated aC74-derived mutant with an altered CDE due to the exchange of 4nucleotides, including 3 of the protected guanine residues. Thisconstruct (C74R1) and the parental (C74) plasmid were analysed in 4independent transfection assays in G₀ versus stimulated (G₂) NIH3T3cells, and in 2 assays comparing G₀ and normally growing cells. FIG. 7Ashows graphic representations of two of these assays, all results arelisted in FIG. 7B. The data clearly indicate that cell cycle regulationin C74R₁ was severely impaired. This loss of regulation was due to adramatically increased activity in G₀ (average 12.8-fold; see FIG. 7B)while expression in G₂ was hardly affected (average 1.6-fold). Takentogether with the protein binding studies, this result strongly suggeststhat the CDE binds a protein or a protein complex that acts as arepressor in G₀/G₁ and is released in the G₂ phase of the cell cycle.

In summary the functional analysis in transient luciferase assays ofvarious truncated cdc25C promoter constructs showed that just 74 bp ofupstream sequence plus 121 bp of transcribed non-translated sequencewere sufficient to confer cell cycle regulation, i.e. induction of thereporter gene around late S/G₂. Experiments currently in progressindicate that a 3′ truncation removing an additional 51 nucleotides hasno influence on the function of the promoter fragment, suggesting thatcell cycle-dependent transcription requires no more than 74 bases ofupstream sequence plus a 50-nucleotide stretch containing the 2initiation sites.

Genomic DMS footprinting revealed the presence of a protein binding sitethat is occupied in a cell cycle dependent fashion directly adjacent 5′to the first initiation site. This element, termed CDE, contains 4 Gresidues in the coding strand at −12, −13, −15 and −16 that areprotected specifically in G₀, but not in G₂. The validity of thisobservation is greatly enhanced by the fact that a G₁-specificprotection pattern was also seen with sorted cells of a normally cyclingpopulation. Since these cells were not synchronised by any means andwere fixed following in vivo exposure to DMS prior to the sortingprocedure, we can largely rule out any experimental artefacts. Wetherefore conclude that the observed cell cycle-regulated proteinbinding to the CDE reflects the physiological situation very closely.

The pattern of protein binding to the CDE raised the possibility thatthis element mediates cell cycle regulation through the interaction witha repressor in G₀/G₁. This hypothesis could be confirmed by functionalassays which showed that mutations in the CDE led to a dramatic increasein reporter gene expression in G₀/G₁, thus largely abolishing cell cycleregulation of the luciferase reporter construct. These results establishthe CDE as a cell cycle-regulated repressor binding site playing a majorrole in the cell cycle-controlled transcription of the cdc25C gene. Thevery poor transcription seen with C20 strongly suggest that the CDE actssolely as a repressor element and is not endowed with any significantenhancer function that could be under cell cycle control. Thisdistinguishes the CDE-binding activity from the transcription factors ofthe E2F/DP family which are repressed in G₁ through their interactionwith the retinoblastoma suppressor protein pRB but act astranscriptional activators later in the cell cycle (for reviews seeNevins 1992; La Thangue 1994).

Further mutagenesis of the CDE (FIG. 12) and the region downstream toposition +30 (FIG. 13) defined the precise position of the CDE tonucleotides −17 to −12 and revealed two additional elements that arecrucial for the proper functioning of the CDE and thus for cell cycleregulation of the cdc25C promoter. Changes in the sequences eitheraround nucleotide positions −6 to −3 the cell cycle homology region(CHR), discussed below, or overlapping the first major initiator Inr atnucleotide positions −2 to +2 led to the same deregulation as mutationof the CDE itself. As shown by in vitro protein binding studies(Barberis et al., 1987) using HeLa cell nuclear extract (Dignam et al.,1983), both elements interact with different proteins. The Inr itselfinteracting with a YY1 complex (Seto et al., 1991), as shown bysupershifts and the binding of recombinant YY1 protein. These resultsstrongly suggest that the function of the CDE-binding protein isdependent on additional interactions with at least one other proteinbinding to the CHR and perhaps the Inr regions and that the CDE-CHRelements have to be contiguous with an Inr. The implication of thisfinding for the construction of chimaeric promoters is that the promoterof the cdc25C gene or other cell cycle regulated promoters must containthe CDE, CHR and the Inr and must be fused to a heterologous enhancer inorder to confer cell cycle regulation on the enhancer. Based on this andusing standard molecular biological techniques, a cdc25C promoterfragment (−20 to +121) was fused to the SV40 early promoter/enhancerregion. The chimaeric promoter obtained exhibited the same cell cycleregulation as the wild type cdc25C gene. See FIG. 14. It would beobvious to one skilled in the art that instead of the SV40 enhancertissue-specific enhancers could be used (Sikora, 1993), which inconjunction with a CDE-CHR-Inr unit would yield chimeric transcriptioncontrol elements that are both cell cycle-regulated and tissue/celltype-specific.

Alignment of the cdc25C CDE-CHR region with the sequences of other knowncell cycle regulated promoters revealed striking similarities in thecase of cyclin A, cyclin F (Kraus et al., 1994), cdc2 (Dalton, 1992) andβ-myb (Lam et al., 1995). Both CDE- and CHR-like elements were found inthese promoters at similar locations relative to the transcription startsites, see FIG. 15. Significantly, any base changes seen in the CDEs inthese promoters were found to be tolerated with respect to cell cycleregulation when introduced into the cdc25C CDE. Results not shown. Inview of these observations we performed genomic footprinting with thecyclin A and cdc2 promoters and found the same situation as in the caseof cdc25C, i.e., the G₀/G₁-specific binding of a protein/protein complexto the CDE. Results not shown. In addition, point mutations largelyabrogated cell cycle regulation, confirming the functional significanceof the CDE-CHR region in these promoters. These observations alsoindicate that the CDE-CHR elements are not totally Inr-specific, sincethe cyclin A, cdc2 and B-myb promoters do not show obvious homologieswith the Inr of the cdc25C gene, but rather dependent on a shortdistance between them and an Inr.

Alignment of the sequences upstream of the CDE also revealed strikingsimilarities in the region of Y_(c)-boxes 1 and 2 (FIG. 16), pointing tocommon targets of repression. Taken together, these data clearly suggestthat CDE-mediated repression is a frequent mechanism of cell cycleregulated transcription, and that the targets of this negativeregulatory mechanism are often the glutamine-rich CCAAT-box bindingproteins (such as NF-1/CTF) and Sp1 family members interacting with therespective enhancers.

The mechanism leading to G₀/G₁ specific repression has been shown to bemediated by a DNA repressor element which is believed to interfere withthe function of the activators interacting with upstream locatedenhancer elements. In the cdc25C gene the sequences upstream of the CDEcontain a Sp1 site and 3 Y_(c)-boxes which act as cis actingtranscriptional activators (FIGS. 5 and 9 to 11). The CDF bindingprotein/protein complex, E-CHR is believed to interfere with thefunction of constitutive, glutamine-rich activators thereby repressingtranscription of the cdc25C gene.

Our observations suggest DNA repressor elements may mediate interferencewith constitutive glutamine-rich activators thereby providing the basisof the periodicity of transcription.

The implication of this finding for the construction of chimaerictissue-specific/cell type-specific, cell cycle regulated promoters isthat tissue-specific enhancers interacting with e.g. glutamine-richactivators are particularly suitable elements to be fused with the DNArepressor element containing cdc25C promoter and other cell cycleregulated promoters operably linked to a DNA repressor element.

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25 21 base pairs nucleic acid single linear DNA unknown 1 GCTGGCGGAAGGTTTGAATG G 21 26 base pairs nucleic acid single linear DNA unknown 2CCCCTCGAGG TCAACTAGAT TGCAGC 26 27 base pairs nucleic acid single linearDNA unknown 3 CGCCCCAACA CTTGCCACGC CGGCAGC 27 26 base pairs nucleicacid single linear DNA unknown 4 CCCCTCGAGG TCAACTAGAT TGCAGC 26 30 basepairs nucleic acid single linear DNA unknown 5 GGTTACTGGG CTGATCAAAGGTTTGAATGG 30 30 base pairs nucleic acid single linear DNA unknown 6CCATTCAAAC CTTTGATCAG CCCAGTAACC 30 17 base pairs nucleic acid singlelinear DNA unknown 7 AGGGAAAGGA GGTAGTT 17 24 base pairs nucleic acidsingle linear DNA unknown 8 TAGATTGCAG CTATGCCTTC CGAC 24 26 base pairsnucleic acid single linear DNA unknown 9 CCTTCCGACT GGGTAGGCCA ACGTCG 26270 base pairs nucleic acid single linear DNA unknown 10 AGGCCTGGGCGCGCGCGGAG ATTGGCTGAC GCAGCTTAGA GGCGAGCGGG GATAGGTTAC 60 TGGGCTGGCGGAAGGTTTGA ATGGTCAACG CCTGCGGCTG TTGATATTCT TGCTCAGAGG 120 CCGTAACTTTGGCCTTCTGC TCAGGGAAGA CTCTGAGTCC GACGTTGGCC TACCCAGTCG 180 GAAGGCAGAGCTGCAATCTA GTTAACTACC TCCTTTCCCC TAGATTTCCT TTCATTCTGC 240 TCAAGTCTTCGCCTGTGTCC GATCCCTACT 270 375 base pairs nucleic acid single linear DNAunknown 11 TTCGTGGGGC TGAGGGAACG AGGAAAACAG AAAGGGTGTG GAGATTGGTGAGAGGGAGAG 60 CCAATGATGC GCCAGGCTCC CCGTGAGGCG GAGSTTACSC SGCAGCCTGCCTAACGCTGG 120 TGGGCCAAAC ACTATSSTGC TCTGGCTATG GGGSGGGGSA AGTCTTACCATTTCCAGAGC 180 AAGCASASGS SSSSAGGTGA TCTGCGAGCC CAACGATAGG CCATGAGGCCCTGGGCGCGC 240 GCGCGGAGAT TGGCTGACGC AGCTTAGAGG CGAGCGGGGA TAGGTTACTGGGCTGGSGGA 300 AGGTTTGAAT GGTCAACGCC TGCGGCTGTT GATATTCTTG CTCAGAGGCCGTAACTTTGG 360 CCTTCTGCTC AGGGA 375 19 base pairs nucleic acid singlelinear DNA unknown 12 GGCTGGCGGA AGGTTTGAA 19 19 base pairs nucleic acidsingle linear DNA unknown 13 TTAGCGCGGT GAGTTTGAA 19 19 base pairsnucleic acid single linear DNA unknown 14 TAGTCGCGGG ATACTTGAA 19 19base pairs nucleic acid single linear DNA unknown 15 AGGGCCGGGTGCGTTTGAA 19 21 base pairs nucleic acid single linear DNA unknown 16GCGCGCGGAG ATTGGCTGAC G 21 21 base pairs nucleic acid single linear DNAunknown 17 CTGGGCTCTG ATTGGCTGCT T 21 21 base pairs nucleic acid singlelinear DNA unknown 18 CTGTCGCCTT GAATGACGTC A 21 17 base pairs nucleicacid single linear DNA unknown 19 GGCGAGCGGG GATAGGT 17 17 base pairsnucleic acid single linear DNA unknown 20 CGGGCTACCC GATTGGT 17 17 basepairs nucleic acid single linear DNA unknown 21 CGAGCGCTTT CATTGGT 17 14base pairs nucleic acid single linear DNA unknown 22 GGGCTGGCGG AAGG 1415 base pairs nucleic acid single linear DNA unknown 23 GCGCGCNNNG ATTGG15 17 base pairs nucleic acid single linear DNA unknown 24 GCGAGCNNNNNGATAGG 17 15 base pairs nucleic acid single linear DNA unknown 25GCGAGCNNNG ATAGG 15

What is claimed is:
 1. A vector for the expression of a desired geneproduct in a cell, comprising a structural gene encoding the desiredgene product operably linked to a chimeric promoter, the promotercomprising a DNA repressor element comprising a sequence selected fromthe group consisting of the sequences TGGCGG, GCGCGG, TCGCGG and GCCGGG,which interacts with a cell cycle specific repressor, a cell cyclehomology region comprising a sequence selected from the group consistingof the sequences GTTTGAA and ACTTGAA, a transcription initiation siteand enhancer elements, in order to regulate gene expression in a cellcycle specific mamner, wherein the promoter and the repressor elementare not naturally present together.
 2. A vector according to claim 1,wherein the promoter is further controlled by a tissue- or celltype-specific regulatory element.
 3. A vector according to claim 1,wherein the DNA repressor element comprises at least part of thesequence 5′-GCTGGCGGAAGGTTTGAATGG-3′ (SEQ ID NO:1) or a mutant orhomologue of SEQ ID NO:1, wherein the DNA repressor element isfunctional to bind the cell specific repressor and wherein the DNArepressor reacts with a cell specific repressor to regulate geneexpression in a cell cycle specific manner.
 4. A vector according toclaim 1, wherein the transcription initiation site is the first majortranscription initiation site of a cdc25C gene.
 5. A vector according toclaim 1, wherein the DNA repressor element is from a cell cycleregulated gene.
 6. A vector according to claim 1, wherein the DNArepressor element is from a cdc25C gene, a cdc2 gene or a cyclin A gene.7. A vector according to claim 1 wherein the gene product is a cytotoxicor cytostatic agent or a prodrug activating enzyme.
 8. A vectoraccording to claim 2, wherein the tissue- or cell type-specificregulatory element is an enhancer.
 9. A vector according to claim 2 orclaim 8, wherein the regulatory element is an enhancer which isactivated by glutamine-rich activators.